Cyclin-dependent kinase 5 (CDK5) regulates the circadian clock
- University of Fribourg, Switzerland
Figures
Figure 1
CDK5 intersects with PER2 and has diurnal activity in the SCN.
(A) Loss of Eap1, Gnd1, or Pho85 compromises growth of PER2-overproducing yeast cells. The yeast mutants eap1∆, gnd1∆, and pho85∆ were identified in a synthetic dosage lethal screen as detailed under Methods. Wild-type (BY4741) as well as eap1∆, gnd1∆, and pho85∆ mutant cells carrying the control plasmid (YCpIF2) or the YPpIF2-mPer2 plasmid (that drives expression of mouse PER2 from a galactose-inducible promoter) were pre-grown on glucose-containing SD-Leu media (to an OD600 of 2.0), spotted (in 10-fold serial dilutions) on raffinose and galactose-containing SD-Raf/Gal-Leu plates, and grown for 3 days at 30°C. (B) Immunoblot was performed on SCN extracts around the clock. SCN from seven animals were pooled at each indicated ZT between ZT0-20. Protein levels of CDK5, CRY1, and HSP90 were analyzed by western blot. (C) Diurnal activity of CDK5 was measured by an in vitro kinase assay. CDK5 was immunoprecipitated at each same time point between ZT0 and ZT20, and half of the immunoprecipitated material was used for performing an in vitro kinase assay using histone H1 (autoradiography, middle panel), whereas the other half was used to quantify the immunoprecipitated CDK5 (upper panel). Coomassie staining shows loading of the substrate (H1). Bottom panel: Quantification of three independent experiments (mean ± SEM). One-way ANOVA with Bonferroni’s post-test, *: p<0.001. (D) The in vitro kinase assay was performed with SCN extracts at ZT12, and either LiCl (GSK3β inhibitor) or 34 μM roscovitine (CDK5 inhibitor). Histone H1 phosphorylation could not be detected with roscovitine treatment, showing the specificity of H1 phosphorylation by CDK5.
Figure 2 with 5 supplements
CDK5 affects the circadian clock.
(A) Wheel-running activity of mice (black bins) infected with AAV expressing scrambled control shRNA, or shCdk5, and an animal with a deletion in the Per2 gene (Per2Brdm1). The actograms are double plotted displaying in one row and below 2 consecutive days. The locomotor activity was confined to the dark period (shaded in gray), while under LD the mice displayed low activity during the light phase (white area). Under DD (continuous gray shaded area) the shCdk5 and Per2Brdm1 animals show earlier onset of activity each day compared with the control animals. The χ2-periodogram analysis for each of the animals is shown below the corresponding actogram to determine the period length (τ). (B) Quantification of the circadian period: 23.3 ± 0.1 hr for the control mice (n = 6, black bar), 22.5 ± 0.2 hr for shCdk5 injected mice (n = 6, gray bar), and 22.4 ± 0.1 hr for Per2Brdm1 mice (n = 4, white bar), (mean ± SEM). One-way ANOVA with Bonferroni’s post-test, **p<0.01. (C) In some cases, mice in which Cdk5 was silenced in the SCN became arrhythmic. (D) Wheel-running activity (black bins) of Per2Brdm1 mice infected with AAV expressing scrambled control shRNA (scr), or shRNA against Cdk5 (shCdk5). The actograms are double plotted displaying in one row and below 2 consecutive days. The dark shaded area indicates darkness during which the free-running period was determined. To the right of each actogram the corresponding χ2-periodogram is shown. The number in each periodogram indicates the period of the animal. (E) Quantification of the circadian period: 22.35 ± 0.03 hr for the scrambled Per2Brdm1 (n = 3, black bar) and 21.77 ± 0.03 hr for the shCdk5 injected Per2Brdm1 mice (n = 5, gray bar). Values are the mean ± SEM, t-test, ***p<0.0001. (F) 1-way ANOVA test on wild-type and Per2Brdm1 animals infected with AAV expressing scrambled control shRNA (scr), or shRNA against Cdk5 (shCdk5). N = 3–6 animals, error bars are the mean ± SEM, Bonferroni multiple comparisons test, ***p<0.001.
Figure 2—figure supplement 1
Characterization of shRNA against Cdk5 Western blot using NIH 3T3 cell extracts transfected with different shRNAs against Cdk5.
All shRNAs were mapped to the Cdk5 sequence. The Western blot reveals that the shRNA D (nucleotides 462 to 490) showed the best silencing activity.
Figure 2—figure supplement 2
Additional activity plots of wild-type mice infected with AAV.
Wheel-running activity of mice infected with AAV expressing scrambled shRNA or shCdk5 and animals with a deletion in the Per2 gene (Per2Brdm1) used for the statistical analysis in Figure 1B and Figure 2—figure supplements 3 and 4.
Figure 2—figure supplement 3
Activity counts per day.
shCdk5 (13878 ± 2877 counts/day, n = 6) and Per2Brdm1 mice (10598 ± 1856 counts/day, n = 4) in DD when compared with the control animals (23478 ± 1277 counts/day, n = 6) as well as in LD conditions: shCdk5 (11894 ± 3379 counts/day, n = 6), Per2Brdm1 mice (11919 ± 1665 counts/day, n = 4) and control animals (24577 ± 2787 counts/day, n = 6).
Figure 2—figure supplement 4
Activity counts in dark or light phase.
Dark: scramble (23276 ± 2817 counts/day, n = 6), shCdk5 (10399 ± 3764 counts/day, n = 6), Per2Brdm1 mice (10521 ± 2052 counts/day, n = 4). Light: scramble (1301 ± 223 counts/day, n = 6), shCdk5 (1495 ± 582 counts/day, n = 6), Per2Brdm1 mice (528 ± 150 counts/day, n = 4). (Mean ± SEM). 1-way ANOVA with Bonferroni post-test, **p<0.01, *p<0.05.
Figure 2—figure supplement 5
Additional activity plots of Per2Brdm1 mice infected with AAV.
Wheel-running activity (black bins) of Per2Brdm1 mice infected with AAV expressing scrambled control shRNA (scr), or shRNA against Cdk5 (shCdk5). The actograms are double plotted displaying in one row and below 2 consecutive days. The dark shaded area indicates darkness during which the free-running period was determined. To the right of each actogram the corresponding χ2-periodogram is shown. The number in each periodogram indicates the period of the animal.
Figure 3 with 4 supplements
Immunohistochemistry in the SCN of control and shCdk5 silenced wild type and Per2Brdm1 mice.
(A) Representative sections of the SCN region after injection of AAVs carrying either scrambled shRNA, or shCdk5. Slices were stained with DAPI (blue), or anti-GFP (green) and anti-CDK5 (red) antibodies. GFP was used as marker for those cells infected by the virus. CDK5 was efficiently down-regulated in the SCN by shCdk5 (red panels) but not by scrambled shRNA, which was as efficiently delivered as shCDK5. As control, the non-infected piriform cortex from the same animal in which Cdk5 was silenced is shown. Scale bar: 200 µm. (B) Analysis of PER2 expression in sections of the SCN of scrambled shRNA, shCdk5 and Per2Brdm1 mice. Silencing of Cdk5 leads to lack of PER2 (red) compared with control at ZT12, which almost resembles the situation observed in Per2Brdm1 animals. Blue color: DAPI staining for cell nuclei. Scale bar: 200 µm.
Figure 3—figure supplement 1
Lower magnification of SCN sections stained for CDK5.
Representative brain sections of normal mice containing the SCN region after injection of AAVs carrying either scrambled shRNA or shCdk5. GFP was used as a marker to illustrate the infected region including the SCN. The CDK5 signal (red) is down regulated in the SCN region of AAV shCdk5 injected brain. Scale bar 500 µm.
Figure 3—figure supplement 2
Quantification of CDK5 signal.
Higher magnification of representative sections of the SCN after AAVs carrying either scrambled shRNA (left column) or shCdk5 (right column). CDK5 if significantly down regulated in brain infected with AAVs expressing shCdk5. Values in the bar diagram represent the mean ± SEM of CDK5 signal relative to the signal in the scramble control, t-test, n = 3, ***p<0.001. Scale bar: 60 µm.
Figure 3—figure supplement 3
Lower magnification of SCN sections stained for PER2.
Representative brain sections of normal mice containing the SCN region after injection of AAVs carrying either scrambled shRNA or shCdk5. GFP was used as a marker to illustrate the infected region including the SCN. As control a SCN section of Per2Brdm1 mouse is shown that was not infected with AAV. The PER2 signal (red) is down regulated in the SCN region of AAV shCdk5 injected brain as it was absent in the Per2Brdm1 SCN. Scale bar 500 µm.
Figure 3—figure supplement 4
Quantification of PER2 signal.
Higher magnification of representative sections of the SCN after AAVs carrying either scrambled shRNA (left column) or shCdk5 (middle column). CDK5 is significantly down regulated in brain infected with AAVs expressing shCdk5. The right column shows a section of Per2Brdm1 mouse not infected with AAVs. Values in the bar diagram represent the mean ± SEM of PER2 signal relative to the signal in the scramble control, t-test, n = 3, **p<0.01. Scale bar: 60 µm.
Figure 4 with 5 supplements
PER2 interacts with CDK5 in a temporal fashion in the cytoplasm.
(A) Overexpression of PER2 and CDK5-HA in NIH 3T3 cells and subsequent immunoprecipitation (IP) using an anti-CDK5 antibody. The left panel shows 5% of the input and the right panel co-precipitation of PER2 with CDK5. (B) Overexpression of PER2-V5 and CDK5-HA in HEK293 cells in presence or absence of 34 μM roscovitine (CDK5 inhibitor) and DMSO (solvent). Left panel shows 5% of the input and the right panel the immunoprecipitation with anti-CDK5 or without antibody. (C) Immunoprecipitation (IP) of PER2 and CDK5 from total mouse brain extract collected at ZT12. Left panel shows the input. The right panel depicts co-immunoprecipitation of PER2 and CDK5 using either anti-CDK5 antibody or anti-GST antibody for precipitation. The middle lane shows PER2-CDK5 co-immunoprecipitation in control animals (Per2+/+) but not in Per2-/- mice illustrating the specificity of the PER2-CDK5 interaction. The * in the blot indicates unspecific signal. (D) Temporal profile of the PER2-CDK5 interaction in total extracts from SCN tissue around the clock. Input was analyzed by immunoblot using anti-CDK5, anti-PER2, and anti-HSP90 antibodies (left panel). CDK5 co-immunoprecipitated PER2 in a diurnal fashion with a peak between ZT12 and ZT16. The statistical analysis of the PER2/CDK5 signal around the clock is shown below (one-way ANOVA with Bonferroni’s post-test, n = 3, *p<0.0001, values are mean ± SEM). * in the blot indicates unspecific signal. (E) Immunoprecipitation of PER2 with CDK5 from cytoplasmic and nuclear brain extracts collected at ZT12. The left panel shows the input and the right panel co-IP of PER2 and CDK5, which occurs only in the cytoplasm but not in the nucleus. The smaller band detected by the anti-PER2 antibody depicts an unspecific band that is smaller than PER2. * in the blot indicates unspecific signal. (F) Slices from the SCN obtained at ZT12 were immunostained with PER2 antibody (green), CDK5 (red), and nuclei were marked with DAPI (blue). Co-localization of the two proteins results in the yellow color. Scale bar: 10 µm. The z-stacks right and below the micrograph confirm co-localization of PER2 and CDK5 (yellow). (G) Purification of the N-terminal half of PER2 (1–576) or the C-terminal half (577–1256) (left panel, coomassie). CDK5-His was pulled down by both recombinant PER2 attached to the glutathione resin, but only the C-terminal was able to retain CDK5 (immunoblot using anti-His antibody, right panel).
Figure 4—figure supplement 1
PER2-CDK5 interaction in HEK cells.
Overexpression of PER2 and CDK5 in HEK 293 cells and subsequent immunoprecipitation (IP) using an anti-CDK5 antibody. The left panel shows the input and the right panel co-precipitation of PER2 with immunoprecipitated CDK5 when both were overexpressed.
Figure 4—figure supplement 2
PER2-CDK5 interaction at different salt concentrations.
Immunoprecipitation (IP) of PER2 and CDK5 from total mouse brain extract collected at ZT12. Left panel shows the input. The middle and right panels depict co-immunoprecipitation of PER2 and CDK5 at two different NaCl concentrations using either anti-CDK5 antibody or anti-PER2 antibody for precipitation. * in the blot indicates unspecific signal.
Figure 4—figure supplement 3
Co-localization of PER2 and CDK5 in SCN tissue.
Temporal profile of the PER2-CDK5 interaction observed by immunofluorescence at ZT0 and ZT12. SCN slices, obtained from mice perfused at ZT 0 and ZT 12, were stained with anti-PER2 antibody (green) and anti-CDK5 antibody (red). Co-localization of the two proteins results in a yellow color, which was observed only at ZT12. Scale bar: 200 µm. The two panels below show a higher magnification depicting single cells in the SCN. The Z-stacks right and below each image show that PER2 and CDK5 mainly co-localize at ZT12. Scale bar: 25 µm.
Figure 4—figure supplement 4
Deletion of PAS domains had no influence on PER2-CDK5 interaction.
NIH 3T3 cells were transfected with vectors carrying PER2-V5, ΔPasA-PER2-V5, or ΔPasB-PER2-V5, and subsequently, immunoprecipitation (IP) using an anti-CDK5 antibody was performed. The results showed that CDK5 was able to interact with all forms of PER2. None of the PAS domains of PER2 seems to be involved in the interaction with CDK5.
Figure 4—figure supplement 5
Scheme of PER2 fragments used for the pull-down assy.
Figure 5 with 6 supplements
CDK5 phosphorylates PER2 at S394.
(A) An in vitro kinase assay was performed using recombinant CDK5/p35 and either GST-PER2 1–576 or GST-PER2 577–1256 as substrate. The samples were subjected to 10% SDS page (Coomassie, left panel) and the phosphorylation of PER2 was detected by autoradiography in order to visualize 32P-labeled proteins (right panel). CDK5 phosphorylates the N-terminal half (1-576) of a GST-PER2 fusion protein whereas the C-terminal half (577–1257) is not phosphorylated. The signal for CDK5/p35 alone indicates CDK5 auto-phosphorylation seen in all lanes when CDK5 is present. (B) Annotated mass spectrum of the tryptic peptide PER2383-397 ILQAGGQPFDYpSPIR containing the phosphorylated residue S394. The red color depicts the y-ion series (1-12) and blue the b-ion series (2–7, a2); y5-98, y8-98, y11-98 show the de-phosphorylated ions. (C) In vitro kinase assay was performed as in (A). The putative phosphorylation site was mutated to aspartic acid (S394D) or glycine (S394G). Both mutations abrogated the CDK5-mediated phosphorylation. Coomassie staining reveals equal expression of the GST-PER2 fragments. The bar diagram at the right shows the quantification of three experiments. One-way-ANOVA with Bonferroni’s post-test, *: p<0.001 (D) The monoclonal antibody produced against P-S394-PER2 does recognizes PER2 (1–576) S394 phosphorylation mediated by CDK5/p35 in presence but not in absence of the kinase or when CDK5 is inactivated by roscovitine. This antibody does not recognize the S394G mutated form even in presence of CDK5/p35. (E) Temporal profile of P-S394-PER2 and total PER2 in SCN tissue. Upper panels show western blots of the corresponding proteins indicated on the right. Below the quantification of three experiments is shown, in which the value at ZT12 of PER2 has been set to 1. The data were double plotted. Values are the mean ± SEM. Two-way ANOVA with Bonferroni’s multiple comparisons revealed that the two curves are significantly different with p<0.0001, F = 93.65, DFn = 1, DFd = 48. (F) Subcellular localization of P-S394-PER2. Total wild-type mouse brain extracts were separated into cytoplasmic (HSP90 positive) and nuclear (laminB positive) fractions. Phosphorylated PER2 was predominantly detected in the cytoplasm with the P-S394-PER2 antibody, whereas the general PER2 antibody detected PER2 in both compartments with higher amounts in the nuclear fraction.
Figure 5—figure supplement 1
Scheme of PER2 fragments used for the in vitro kinase assay.
The fragment 1–576 covers the sites that might be phosphorylated by CDK5 on the basis of the conserved consensus (S/T)PX(K/H/R).
Figure 5—figure supplement 2
Additional controls for in vitro kinase assay.
An in vitro kinase assay performed in presence of recombinant CDK5/p35 and using as substrate the GST-PER2 1–576.
Figure 5—figure supplement 3
Testing specificity of the in vitro kinase assay.
The reactions were treated either with LiCl (inhibitor of GSK3β kinase activity) or roscovitine (inhibitor of CDK5 kinase activity) in order to highlight the specificity of the PER2 phosphorylation mediated by CDK5.
Figure 5—figure supplement 4
Characterization of antisera against P-S394-PER2.
Different antisera against the PER2 peptide sequence FDY{pSer}PIRFRTRNGEC were tested by in vitro kinase assay using recombinant GST-PER2 1–576 (in presence or absence of CDK5/p35) followed by WB. Even if at this stage, it was necessary to choose an antiserum that recognized the PER2 peptide regardless of its phosphorylation status, the antiserum one was able to discriminate the two forms and was therefore used for the following amplifications.
Figure 5—figure supplement 5
Characterization of hybridomas against P-S394-PER2.
Different hybridomas producing antibodies against the PER2 peptide sequence FDY{pSer}PIRFRTRNGEC were tested by in vitro kinase assay using recombinant GST PER2 1–576 (in presence or absence of CDK5/p35) followed by WB. From 16 different clones, the positive ones are shown. The clone 10E12 was used to produce the final antibody.
Figure 5—figure supplement 6
Validation of anti-P-S394-PER2 antibody.
Total protein extracts were obtained from wild-type, Per2Brdm1 and Per2-/- mouse brains at ZT12. Western blot was performed in order to validate the specificity of the antibody against the phosphorylated serine 394 on PER2. Only samples obtained from WT tissues showed the phosphorylated form of the protein. Antibody against total PER2 was used as control, which, positively detected the protein only in extracts obtained from wild-type mice.
Figure 6 with 4 supplements
CDK5 affects PER2 stability and nuclear localization.
(A) Western blot of NIH 3T3 cell extracts with and without roscovitine treatment. When roscovitine inhibited CDK5, less PER2 protein was detected in cell extracts. The bar diagram below shows values (mean ± SEM) of three experiments with significant differences between roscovitine treated and untreated cells, t-test, *p<0.001. (B) CRISPR/Cas9-mediated knockout of Cdk5 in NIH 3T3 cells. Western blot shows absence of PER2 in cells when Cdk5 is deleted. (C) Titration of CDK5 knock-down as revealed by Western blotting. PER2 levels decreased proportionally to increasing amounts of shCdk5. 2.7 μM of shCdk5 (red) was used for subsequent experiments. The value without shCdk5 was set to 1. One-way ANOVA with Bonferroni post-test, n = 4, ***p<0.001, ****p<0.0001, mean ± SD. The * in the blot indicates unspecific signal. (D) Temporal profile of protein abundance in NIH 3T3 cells 0, 3 and 6 hr after inhibition of protein synthesis by 100 μM cycloheximide (CHX) in presence of scrambled shRNA, or shCdk5, respectively (2.7 μM of the respective shRNA was used). The diagram below shows quantification of PER2 protein over time. Linear regression with 95% confidence intervals (hatched lines) indicates that knock-down of Cdk5 leads to less stable PER2 (shCdk5 t1/2=4h, scr t1/2=11h). Two-way ANOVA with Bonferroni’s post-test revealed that the two curves are significantly different, n = 3, p<0.01, F = 24.53, DFn = 1, DFd = 4. (E) Inhibition of the proteasome by epoxomycin in cells with shCdk5 leads to amounts of PER2 that are higher compared with the levels without epoxomycin treatment and are comparable to the levels observed in cells without Cdk5 knockdown. Diagram below displays the quantification of three experiments. Scrambled shRNA values were set to 1. One-way ANOVA with Bonferroni’s post-test shows no significant reduction of PER2 in shCdk5 cells in presence of epoxomycin, but significantly lower values in absence of epoxomycin when compared with scrambled shRNA treatment. One-way ANOVA with Bonferroni’s post-test, n = 3, p<0.001. (F) PER2 abundance in nuclear extracts of NIH 3T3 cells. Knockdown of Cdk5 reduces PER2 levels in the nucleus as revealed by Western blotting. HSP90 = cytosolic marker, LaminB = nuclear maker. (G) Immunofluorescence of PER2 (red) at ZT12 in mouse SCN sections after infection with AAV (green) expressing scrambled shRNA (left panel), or shCdk5 (right panel). Nuclei are visualized by DAPI staining (blue). PER2 can only be observed in the nucleus in presence (white arrow heads) but not in absence of CDK5 (white arrow). Scale bar = 7.5 µm. (H) Co-immunoprecipitation of CRY1 by PER2 in NIH 3T3 cells. Substitution of S394 to G in PER2 reduces the levels of co-precipitated CRY1 (right panel). The left panel shows the input. The bar diagram on the right displays the quantification of three experiments, where the amount of precipitated CRY1 by PER2 is set to 1. Paired t-test reveals a significant difference between the amounts of CRY1 precipitated by PER2 and the S394G PER2 mutation, n = 3, *p<0.05, mean ± SD.
Figure 6—figure supplement 1
Characterization of Cdk5 ko cell morphology.
NIH 3T3 and CRISPR/Cas9 Cdk5-deficient cells were photographed using a bright light microscope (100 x). A clear difference in shape and thickness between the two cell lines could be observed. CRISPR/Cas9 Cdk5 cells appeared rather stressed and not to be dividing well.
Figure 6—figure supplement 2
Selection for absence of Cdk5 mRNA.
PCR to detect the mutation of the genomic Cdk5 DNA sequence was performed on different putative knock-out clones. Among these, clones 3 and 5 showed the Cdk5 PCR product, demonstrating that showed they were false positive for knocking out the gene. A positive control (WT genomic DNA) and negative control (water as template) were used.
Figure 6—figure supplement 3
Selection for absence of CDK5 protein.
Total protein extracts were obtained from clone 1, 10 and WT NIH 3T3. Western blot was performed in order to verify which clone no longer expressed CDK5. Clone number 10 was confirmed to be a positive CRISPR/Cas9 Cdk5 knock-out clone.
Figure 6—figure supplement 4
Additional examples of cellular localization of PER2.
Additional examples of immunofluorescence of PER2 (red) at ZT12 in mouse SCN sections after infection with AAV (green) expressing scrambled shRNA (left column of panels), or shCdk5 (right column of panels). Nuclei are visualized by DAPI staining (blue). PER2 can only be observed in the nucleus in presence but not in absence of CDK5. Scale bar = 2 µm.
Figure 7
Model illustrating the regulation of PER2 by CDK5.
The upper row illustrates phosphorylation of PER2 at S394 by CDK5 that subsequently favors interaction with CRY1 and leads to transport into the nucleus, where the PER2/CRY1 complex inhibits BMAL1/NPAS2 (or in the periphery CLOCK)-driven transcriptional activation. Of note is that CDK5 potentially phosphorylates other clock relevant components, such as CLOCK, PER1 and CKI. The lower part illustrates that inhibition of CDK5 leads to a lack of S394 PER2 phosphorylation, which renders the PER2 protein more prone to degradation by the proteasome. CRY1 does not form a complex with PER2 and hence PER2 is not transported into the nucleus. CRY1 enters the nucleus independently and can inhibit the BMAL1:NPAS2 (or in the periphery CLOCK) transcriptional complex. This model is consistent with the dual modulation of transcriptional inhibition (Ye et al., 2014; Xu et al., 2015). Transcriptional inhibition is modulated in an intricate unknown manner by various additional factors (gray) (Aryal et al., 2017) that may be cell type specific.
Figure 8
Workflow of the in vitro kinase assay.
Workflow of the in vitro kinase assay performed using immunoprecipitated CDK5 from SCN protein extracts is schematized here. Seven mice were sacrificed, SCN tissues were isolated and pooled together every 4 hr starting from ZT 0 (lights on) until ZT20 (ZT12 lights off). Total protein was obtained from each pool of tissues, the quality of the extracts was checked by WB, and subsequently CDK5 was immunoprecipitated at each time point. Agarose beads detained the immunoprecipitation and one half of the precipitate was used for an in vitro kinase assay using as substrate commercial histone H1 as substrate. The other half was analyzed by WB in order to quantify the amount of protein immunoprecipitated, which was used for the kinase assay. Kinase activity around the clock was quantified using the following formula: (32P-H1/total H1)/amount of immunoprecipitated CDK5.
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Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Genetic reagent (M. musculus) | Per2Brdm1 | Jackson Laboratory | Stock #: 003819 | PMID: 10408444 |
| Genetic reagent (M. musculus) | B6;129P2-Per2tm1Ual/Biat | European mouse mutant archive | Strain ID EM:10599 | PMID: 26838474 |
| Cell line (M. musculus) | NIH3T3 | ATCC | Cat. #: ATCCRCRL-1658Tm | Immortalized Mouse fibroblast cells |
| Cell line (M. musculus) | NIH3T3 CRISPR/Cas9 Cdk5 KO | Origene | Cat. #: KN303042 | Immortalized Mouse fibroblast cells.7 ug/ml of puromycin are required for cells propagation |
| Cell line (Human) | HEK | ATCC | Immortalized Kidney fibroblast cells | |
| Transfected construct (M. musculus) | Sh RNA CDK5 plasmids | Origene | Cat. #: TL515615 A/B/C/D | |
| Transfected construct (M. musculus) | Sh RNA scramble | Origene | Cat. #: TR30021 | |
| Antibody | anti-PER2-1 (Rabbit polyclonal) | Alpha Diagnostic Lot # 869900A1.2-L | Cat. #: PER21-A RRID: AB_2236875: | 1:200 (IF) 1:50 (IP) 1:500/1:1000 (WB) |
| Antibody | anti-Cdk5 clone 2H6 (Mouse monoclonal) | Origene Lot # A001 | Cat. #: CF500397 RRID: AB_229166 | 1:20 (IF) 1:50 (IP) 1:500/1:1000 (WB) |
| Antibody | anti-GFP (Rabbit polyclonal) | Abcam | Cat. #: ab6556 RRID: AB_305564 | 1:500 (IF) |
| Antibody | anti-rabbit IgG (H+L) (Donkey polyclonal) | Alexa Fluor 488 Lot # 132876 | Cat. #: 711-545-152 RRID: AB_2313584 | 1:500 (IF) |
| Antibody | anti-mouse IgG (H+L) (Donkey polyclonal) | Alexa Fluor 647 Lot # 131725 | Cat. #: 715-605-150 RRID: AB_2340862 | 1:500 (IF) |
| Antibody | anti-rabbit IgG (H+L) (Donkey polyclonal) | Alexa Fluor 647 Lot # 136317 | Cat. #: 711-602-152 | 1:500 (IF) |
| Antibody | anti-HA (Mouse monoclonal) | Roche | Cat. #: 11583816001 RRID: AB_2532070 | 1:1000 (WB) |
| Antibody | anti-GST (Mouse monoclonal) | Sigma | Cat. #: G1160 RRID: AB_259845 | 1:1000 (WB) |
| Antibody | PER2 Phosphor Serine 133 (mouse monoclonal) | GenScript Company | Provided by the corresponding author | WB: 1:200 |
| Other | DAPI | Termofisher | Cat. #: D3571 RRID: AB_2307445 | (1 µg/mL) |
| Recombinant DNA reagent | Supplemental Table II | Complete list provided in the paper | ||
| Commercial assay or kit | pCR2.1-TOPO cloning | Thermofisher | Cat. #: K4500-01 | |
| Commercial assay or kit | QuikChange Site-Directed Mutagenesis Kit | Agilent | Cat. #: 200518 | |
| Chemical compound | Polyethylenimine, Linear, MW 25000, Transfection Grade (PEI 25K) | Polyscience Europe | Cat. #: 23966–1 | |
| Chemical compound | Roscovitine | Merk | Cat. #: R7772-1MG | |
| Chemical compound | Protein Agarose Beads | Roche | Cat. # 11 719 408 001 | |
| Chemical compound | cOmplete, EDTA-free Protease Inhibitor Cocktail | Merk | Cat. # 11873580001 | |
| Chemical compound | Isopropyl β-D-1-thiogalactopyranosid | Sigma-Aldrich | Cat. # 367-93-1 | |
| Chemical compound | L-Glutathione reduced | Merk | Cat. # 70-18-8 | |
| Chemical compound | Cycloheximide | Merk | Cat. # | |
| Chemical compound | Epoxomicin | Sigma-Aldrich | Cat. # | |
| Peptide, recombinant protein | Cdk5/p35 Protein, active, 10 µg | Millipore | Cat. # 14–477 | |
| Peptide, recombinant protein | Histone H1 | Sigma-Aldrich | Cat. # H1917-100UG | |
| Software | Prism | GraphPad | Version 8.2.0 | |
| Software | ImageJ | ImageJ | Version 1.49 RRID: SCR_00370 | |
| Software | ClockLab | Actimetrics | Acquistion version: 3.208 Analysis version: 6.0.36 RRID: SCR_0114309 | |
| Software | Leica application Suite Advanced Fluorescence | Leica | Version 2.7.3.9723 |
Additional files
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Supplementary file 1
Phosphorylation sites of GST-Per2 (1-576) detected by mass spectrometry.
The serine at position 394 stands out as the best localized phosphorylation site within a CDK5 consensus motif with a high peptide score (highlighted in yellow). The colored diagram shows the structural elements of PER2 (1–576) with the S394 phosphorylation site indicated. PEP: posterior error probability; Loc. Prob.; localization probability.
- https://cdn.elifesciences.org/articles/50925/elife-50925-supp1-v2.xlsx
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Supplementary file 2
Plasmids.
- https://cdn.elifesciences.org/articles/50925/elife-50925-supp2-v2.docx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/50925/elife-50925-transrepform-v2.docx
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Cyclin-dependent kinase 5 (CDK5) regulates the circadian clock
eLife 8:e50925.
https://doi.org/10.7554/eLife.50925
